Mm0 202n

For KD, OE or KD + OE experiments, 5–10  pl of ROMO1 dsRNA, mRNA or dsRNA + mRNA was microinjected into the cytoplasm of a parthenogenetically activated oocyte respectively, using an Eppendorf Femto-Jet (Eppendorf, Hamburg, Germany) and a Nikon Diaphot Eclipse TE300 inverted microscope (Nikon, Tokyo, Japan) equipped with a Narishige MM0‐202N hydraulic 3-dimensional micromanipulator (Narishige, Amityville, NY, USA). After injection, oocytes were cultured in PZM‐5 medium. The control group was microinjected with green fluorescent protein dsRNA or empty plasmid.

Mitochondria were prepared by differential centrifugation. The ADSCs were prepared with HTF at 10⁷ cells per ml. Then the cells were treated 4-6 times with a cell homogenizer on ice. The homogenate was centrifuged for 20 min at 2000r/m, the supernatant was collected and centrifuged for 20 min at 10000r/m again. The sediments were resuspended very slowly with 1μL HTF. That is, the mitochondria extracted from 10⁷ ADSCs were in 1uL HTF solution. All the processes were carried out at 4°C, and the extracts were maintained at 4°C for microinjection. For the GV oocytes, 7-8pL mitochondrial in HTF were micro-injected into each oocyte using a Nikon Diaphot ECLIPSE TE 300 (Nikon UK Ltd., UK) inverted microscope equipped with Narishige MM0202N hydraulic three-dimensional micromanipulator (Narishige Inc., USA) and microinjection was completed within 30 minutes. For MII oocytes, 10pL mitochondria were microinjected into each oocyte combing ICSI (for details, please see “ICSI and embryo transfer” below).

Microinjection of Rac3 SiRNAs (Gene Pharma, Shanghai, China) or Alexa 488-phalloidin (Invitrogen, Carlsbad, CA, USA) into GV oocytes was performed using a Nikon Diaphot ECLIPSE TE 300 (Nikon UK Ltd., Kingston upon Thames, Surrey, UK) inverted microscope equipped with Narishige MM0202N hydraulic three-dimensional micromanipulators (Narishige Inc., Sea Cliff, NY, USA) and completed within 30 minutes.
Rac3 SiRNAs were microinjected into GV oocyte to knockdown Rac3. The subsequent sequences of Rac3 siRNAs were used at 20 μM each, Rac3 siRNA-1: 5’- GGAAGACUACGAUAGGCUUTT-3’;
Rac3 siRNA-2:5’- GCAAGAAGUGCACUGUAUUTT-3’;
Rac3 siRNA-3: 5’—GCCUUCCCAGGAGAAUAUATT-3’.
The same amount of negative control siRNAs were injected as control. Microinjected oocytes were cultured in M16 medium with 100μM IBMhX for 24 h. Then the oocytes were cultured in M16 medium.
For live oocyte imaging, after 1–2 hours of culture, the microinjected oocytes cultured in M16 medium with 10ng/mL Hoechst 33342 were used for live oocyte imaging on a Perkin Elmer precisely Ultra VIEW VOX confocal Imaging System (PerkinElmer, Waltham, MA, USA).

For knock‐down experiments, GSNOR dsRNA was microinjected into the cytoplasm of a parthenogenetically activated oocyte using an Eppendorf Femto‐Jet (Eppendorf AG, Hamburg, Germany) and Nikon Diaphot ECLIPSE TE300 inverted microscope (Tokyo, Japan) equipped with a Narishige MM0‐202N hydraulic three‐dimensional micromanipulator (Narishige, Inc, Sea Cliff, NY, USA). After injection, oocytes were cultured in PZM‐5. The control group was microinjected with enhanced green fluorescent protein (EGFP) dsRNA.

Fully grown GV stage oocytes were microinjected with aliquots of 5–10 pL of 50 μM KIF2A siRNA (5′-GGA UGU UGA UGC UAC AAA UTT-3′, 5′-AUU UGU AGC AUC AAC AUC CTT-3′) (GenePharma, Shanghai, P. R. China) or negative control siRNA (5′-UUC UCC GAA CGU GUC ACG UTT-3′, 5′-ACG UGA CAC GUU CGG AGA ATT-3′) (GenePharma, Shanghai, P. R. China). The process was performed with a Narishige MM0-202N hydraulic three-dimensional micromanipulator (Narishige Inc., Sea Cliff, NY, USA) under the Nikon Diaphot ECLIPSE TE300 inverted microscope (Nikon UK Ltd., Kingston upon Thames, Surrey, UK). The oocytes were cultured for 24 h in M2 medium with 2.5 μM milrinone after microinjection. After three 2-min washes in fresh M2 medium, the oocytes were then cultured in fresh M2 medium under mineral oil in a 5% CO₂ incubator, maintaining at 37 °C in humidified air. Oocytes were collected at different stages for further analysis.

Microinjection of JMY dsRNA into the cytoplasm of oocytes was performed as described previously [18] (link) with the Femtojet constant flow system (Eppendorf AG, Hamburg, Germany) and a Nikon Diaphot ECLIPSE TE300 inverted microscope (Nikon UK Ltd., Kingston upon Thames, Surrey, UK) equipped with a Narishige MM0-202N hydraulic three-dimensional micromanipulator (Narishige Inc., Sea Cliff, NY, USA). Each oocyte was injected with approximately 10 pL (1 µg/uL) of JMY dsRNA, and oocytes were cultured under paraffin oil at 38.5°C. The developmental stage of oocytes was determined by staining with 1 µg/mL of 4′-6-diamidino-2-phenylindole (DAPI) for 10 min. The control oocytes were microinjected with 5–10 picoliter of green fluorescent protein dsRNA. All microinjection experiments were performed at least five independent times, and approximately 100 oocytes were injected in each group.

Microinjections were completed within 1 h using an Eppendorf microinjector and a Nikon Diaphot ECLIPSE TE300 inverted microscope (Nikon U.K. Ltd.) equipped with a Narishige MM0-202N hydraulic three-dimensional micromanipulator (Narishige Inc.). To deplete BECN1 in germinal vesicle (GV) oocytes, 10 pl (1 μg/μl) of dsRNA was microinjected into the cytoplasm of the oocytes. After injection, oocytes were cultured for 24 h in TCM-199 medium containing 1 mM dbcAMP. Oocytes were then transferred to fresh TCM-199 medium and cultured for 48 h. Control oocytes were microinjected with 10 pl of water.